High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Abstract

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 ؋ 10 ؊8 to 2 ؋ 10 ؊6 , 1 ؋ 10 ؊8 to 4 ؋ 10 ؊8 , and <4 ؋ 10 ؊9 to 4 ؋ 10 ؊8 per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 ؋ 10 ؊4 to 1 ؋ 10 ؊3 , 9 ؋ 10 ؊4 to 3 ؋ 10 ؊3 , and 5 ؋ 10 ؊4 to 4 ؋ 10 ؊3 for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.Recent nucleotide and whole-genome analyses have revealed that most bacterial genomes contain large amounts of bacteriophage DNA (18). This finding suggests that lateral gene transfer by bacteriophages has contributed significantly to the acquisition of new genetic traits, the ability of bacteria to exploit new environments, and the genetic diversity of bacteria (27). Since bacteriophage-mediated gene transfer was first recognized (32), transduction has been found to occur in many phage-host systems, and various aspects of transduction, including molecular mechanisms, physiologic and genetic characterization of transductants, and ideal environments for transduction, have been investigated (29).For nearly a half-century, culture methods using selective agar media have played a leading role in the study of gene transfer (5). Genetic characteristics such as amino acid deficiency repair and antibiotic resistance have been used for selection of transductants (9). Transduction frequencies were shown to differ over orders of magnitude from 10 Ϫ11 to 10 Ϫ5 per bacteriophage, which are lower than those for conjugation and transformation (14, 27). However, current knowledge of horizontal gene transfer via bacteriophages in the environment is rather limited because of methodological constraints. Conventional methods for the detection of gene transfer depend on high levels of gene expression and culturability on selective media. Although these methods have led to an understanding of the genetic and physiologic characteristics of transductants and the molecular mechanism of transduction, they have limited abilities to quantify the genetic material introduced into individua...

Ecological and Evolutionary Benefits of Temperate Phage: What Does or Doesn't Kill You Makes You Stronger

“…While all phages are likely to have the potential for transduction through errors in excision or DNA packaging here is significant variation in transduction rate between phages. [14] Phages employing the "headful" DNA packaging mechanism for example have very high rates of transduction [15,16] while it is uncommon in others. [17] For the transducing phage there can be serious costs, particularly if some or all of the phage's own genome is excluded from the virus capsid.…”

Persistence of Escherichia coli Clones and Phenotypic and Genotypic Antibiotic Resistance in Recurrent Urinary Tract Infections in Childhood

“…However, the role of the failure of antimicrobial therapy, due to initial or changed antimicrobial resistance after a horizontal transfer of genes or gene cassettes (26,29), in causing the persistence of E. coli in the urinary tract is not well known. Also, no association between the presence of integrons and sul genes, in either clonal relapsing or recurrent individual E. coli strains, and antibiotic therapy has been established yet.…”

Transfer of a Phage T4 Gene into Enterobacteriaceae , Determined at the Single-Cell Level

“…CPRINS uses one primer and results in linear amplification of the target DNA inside cells, and multiply labeled fluorescent probe sets are applied for detection of the amplicons to improve the specificity and sensitivity of CPRINS (3). Previously, CPRINS-FISH did clarify the movement of DNA of a specific gene among Escherichia coli cells at the single-cell level (4).…”

“…After amplification, filters were rinsed with 0.1% Nonidet P-40 and sterile deionized water, dehydrated in 99% ethanol, and vacuum dried. Hybridization with Alexa Fluor 546-labeled polynucleotide probes (T4-tail2664, T4-tail2720, T4-tail2769, T4-tail2818, T4-tail2869, and T4-tail2922), washing, and DAPI staining were performed as described in a previous study (4). In order to exclude the possibility of nonspecific probe binding to cell structures other than the target DNA in the target cells, FISH using laboratory strains without amplification of target DNA and CPRINS-FISH targeting the tail fiber genes in E. coli strains that did not carry the genes were performed.…”

“…CPRINS-FISH targeting the tail fiber gene of phage T4GT7 was performed as described by Kenzaka et al (3,4), except for the probe/primer sequences and thermal conditions. After cell wall permeabilization by lysozyme treatment (3), the CPRINS reaction was performed under the following conditions: a hot start at 95°C for 9 min, denaturation at 94°C for 1 min, annealing at 60°C for 30 s, and extension at 72°C for 1.5 min for primer T4-tail2983r.…”